Tumor hypoxia is an important characteristic of solid tumors and a modulator of therapeutic response. Because oxygen diffuses 100-150 ?m from blood vessels, fast growing tumors often have two hypoxic regions: a chronically hypoxic region in the center of the tumor and a cycling (or acute) hypoxic region within the diffusion distance. Numerous studies have found that hypoxia, especially its temporal fluctuation, leads to enhanced tumor metastasis and resistance to radiation and drugs. Strategies to reduce hypoxia by increasing delivery of oxygen and decreasing oxygen consumption within the tumor are both being explored to overcome hypoxia- induced resistance to radiation and drugs. Therefore, there is growing demand for technologies that noninvasively measure tumor oxygenation temporally in vivo to enable advances in drug screening, development and optimization. However, the relative contributions of chronic hypoxia and cycling hypoxia (CH) as well as the therapeutic responses are difficult to determine primarily due to the lack of a noninvasive tool to continuously quantify the temporal profile of tumor hypoxia in vivo. We have recently developed a side-firing fiber optic sensor and a 1st generation (GEN-1) frequency-domain near-infrared spectroscopy (FD-NIRS) for quantification of tissue oxygenation and total hemoglobin content in model tumors. The flat sensor can be easily and reliably attached to a tumor surface and thus is an ideal tool for longitudinal monitoring of rodent tumor models and studying anti-hypoxia drugs in vivo. The objective of the proposed project is to develop a 2nd generation (GEN-2) FD-NIRS instrument with improved speed and throughputs and validate it for longitudinal assessment of tumor hypoxia and the efficacy of chemoradiotherapy. We hypothesize that 1) the temporal profiles of hypoxia vary in different breast tumors and 2) biguanide drugs (e.g., metformin or phenformin) can reduce the oxygen consumption of breast cancer cells, thus improving their radiosensitivity. The following specific aims will be conducted to test the hypotheses: (1) to construct a 2nd generation FD-NIRS instrument with 10x speed and 15-dB better throughputs; (2) to quantify the characteristics of tumor hypoxia in orthotopic models of breast cancer; and (3) to assess the efficacy of metformin and irradiation in orthotopic models of breast cancer using the GEN-2 device. Our long- term goal is to develop a portable/wearable, low-cost FD-NIRS device that can aid in development and optimization of anti-hypoxia drugs and chemoradiotherapy. The successful completion of the aims will not only generate new knowledge about tumor hypoxia and lay the foundation for subsequent clinical studies to further delineate the role of hypoxia in cancer therapy, but also will enhance the biophotonics research and education at The University of Akron.